Project Summary The long-term goals of this research are to define the mechanisms by which foreign viral or plasmid DNA is recognized or sensed in mammalian cells and then epigenetically silenced. Herpes simplex virus (HSV) genomic DNA in the virion is not associated with histones, but the host cell rapidly adds heterochromatin to the HSV genome upon entry into the nucleus. The cellular factors that sense the foreign viral DNA are poorly characterized. We had shown previously that the host IFI16 protein senses HSV DNA and stimulates an innate response, and during the previous funding period we showed that IFI16 promotes epigenetic silencing of ICP0-null HSV. IFI16 is the only host protein known to increase heterochromatin marks on HSV chromatin. The ND10 proteins PML, Sp100, Daxx and ATRX have all been reported to restrict ICP0-null mutant virus replication and gene expression, but no information is available about how they affect HSV chromatin. We have designed a novel quantitative imaging analysis tool for detection of input viral DNA and host factors, and we have exciting new results showing the sequential association of IFI16 with viral input DNA at 30 minutes postinfection (pi) and ATRX with viral input DNA at 0.5-2 hour pi. The associations of IFI16 and ATRX with input viral DNA appear to be independent and their effect on viral replication is additive. Because we have shown that the viral genome is loaded with heterochromatin by 1-2 hours pi, IFI16 and/or ATRX are strong candidates for a role in the initial sensing of viral DNA and loading of heterochromatin. We also have used a novel small molecule screen to identify molecules that inhibit ICP0-specific transactivation that will provide important probes of ICP0 function. In this application, our specific aims are to 1. Determine the role and mechanisms involving IFI16 in early HSV DNA sensing and chromatinization. 2. Determine the role and mechanisms involving ATRX and other ND10 proteins in early HSV DNA chromatinization. 3. Define the functional mechanisms of ICP0 action on host DNA sensors by determining the mechanisms by which ICP0 promotes degradation of IFI16 and by studies of the mechanism of action of small molecules inhibiting ICP0-dependent gene expression. These studies will provide important new basic knowledge of the mechanisms of sensing of foreign DNA and enable new therapeutics for the herpesviruses and improved gene delivery mechanisms using viral and DNA-based technology.